/ Int. J. Agric. Biol., Vol. 13, No. 3, 2011
components in the medium, carbon source plays a criticalrole as sources of precursors and energies for synthesis of biomass building blocks and secondary metabolite production (Wang
2008 & 2010; Jia
2009).Therefore, influences of medium components andenvironmental conditions are an initial and important step toimprove metabolite production of the genus
.This research work is concerned with identify of thestrain KGG32 based on morphological, physiological, biochemical characteristics and determine the effects of various environmental factors on antimicrobial metabolite production and cell growth, under fermentation conditions.
MATERIALS AND METHODS
Microorganisms, media, and growth conditions:
sp., KGG32 was used in this investigation.The strain was isolated from the soil sample collected fromGeçitköy Pond, North Cyprus as described previously(Oskay
2010). Spore and mycelia suspensions of
sp., KGG32 was maintained in sterile 15%glycerol deposited at –20°C in culture collection of BiologyDepartment, Celal Bayar University, Manisa Turkey.Target Microorganisms: Different test microorganisms(15) were used throughout this study (Table I). Cultures of test bacteria were grown in Mueller-Hinton Broth (Oxoid) at37
C for 24 h and stored in nutrient agar slants at 4
C.Yeasts were cultured on yeast extract malt extract broth at30
C for 48 h and they maintained on the potato dextroseagar (PDA, Oxoid). The bacteria were obtained from theDepartment of Biology, Ege University (
Characterization of the
sp., KGG32Cultural and phenotypic characteristics:
A set of morphological and physiological characteristics of the strainwere examined after incubation for 7-14 days at 28
C onvarious media described by the International
Project (ISP) (Shirling & Gottlieb, 1966) and the Bergey’sManual of Systematic Bacteriology (Williams
1983a;Cross, 1989). Mature aerial mycelium and substratemycelium pigmentation were recorded on ISP5, aerial masscolor on ISP3 and ISP4, melanin production on ISP6 andISP7 following incubation at 27
C for 21 days using areference color key (Prauser, 1964). For morphology of spore bearing hyphae with entire spore chain the strain wasgrown on different ISP media (ISP 3–6) and observed witha light microscope using cover-slip method. Temperaturerange, pH range, and NaCI tolerance for growth wererecorded on Bennet’s agar plates that were incubated at27
C for up to 21 days. Resistance to antibiotics wasestimated by the conventional paper disc agar diffusion bioassay. Utilization of carbohydrates was investigated onISP9 medium using glucose as positive control. The abilityto utilize nitrogen sources was determined in a basalmedium containing glucose 10 g, MgSO
O 0.5 g,FeSO
O 0.01 g, K
1.0 g, NaCI 0.5 g, agar 3.0 gand distilled water 200 mL; after 15 days.
Other physiological and biochemical characteristics weredetermined using the method described by Williams
(1983a & b). All tests were performed at 27
Analysis of the whole-celldiaminopimelic acid isomers (
-DAP) andthe sugars was done by the method of Lechevalier andLechevalier (1970) using the thin layer chromatography(silica gel plates (20X20, 60 F
, Merck, Darmstadt,Germany).
Selection of suitable culture conditions for the optimumproduction of antimicrobial metabolite:
Bioactivemetabolite production of the strain was optimized by usingdifferent cultural parameters such as pH, temperature,carbon and nitrogen sources and time incubation in hours.
Effect of carbon and nitrogen sources:
The influence of different carbon (glucose, glycerol, starch, maltose &sucrose) and nitrogen sources (meat extract, yeast extract, peptone, tryptone & L-asparagine) were studied tostandardize the antibiotic production. The carbon andnitrogen sources were added to the pre-optimized medium before sterilization at a concentration of 1% (w/v) and 0.5%(w/v), respectively. The carbon and nitrogen sourcesupporting the maximum production of metabolite wereselected for the further studies. The temperature and pHwere set to 30
C and at 7.5, respectively.
Effect of initial pH of the culture medium:
To determine,influence of initial pH value of culture medium on growthand bioactive metabolite production; the strain wascultivated in the medium with different initial pH values (6– 9). The pH was adjusted using 0.1 N hydrochloric acid or 0.1 N sodium hydroxide. The initial pH of culture mediumachieved maximum antibiotic production was used for subsequent study.
Effect of incubation temperature:
The optimumtemperature for cell growth and bioactive metabolite yieldwas assayed by incubating the production medium atdifferent temperatures varying from 20-37
C, maintainingall other conditions at optimum levels at originalconcentration.
Preparation of Fermentation medium:
For maximum production of antimicrobial metabolite from the
sp. KGG32; was inoculated in ISP2 mediumthat was used for development of inoculums. The seedculture was conducted in 250 mL Erlenmeyer flaskscontaining 40 mL of medium (same with fermentationmedium) by inoculating 2.0 mL of spore suspensioncontaining 1.0 × 10
spores/mL and cultivated under agitation (180 rpm) at 30
C for 2 days. After optimization of the fermentation parameters, a sample of 2 mL (5%, v/v) of spore suspension containing 1.0 × 10
spores/mL wasinoculated to 200 mL of into the desired medium containingcarbon source 10.0 g, meat extract 3.0 g, yeast extract 1.0 g, bacteriologic peptone 5.0 g, CaCO
1.0 g, K
HPO4 1.0 g,KH
1.0 g, trace element solution (Shirling & Gottlieb,1966)
1 mL and distilled water 1000 mL. The initial pH wasadjusted to 7.5 with 0.1 N NaOH or 0.1 N HCI as required